Research on the binding media used in ancient tomb mural paintings is a hot topic in international scientific archaeology. In this study, the Enzyme-Linked ImmunoSorbent Assay (ELISA) technique was applied to analyze and determine the concentration of dog collagen in the red, black and yellow mural painting samples in the tombs built in 5th century in North China. It is found that the dog collagen concentration in the dry red mural painting samples was 7.328mg/L, but no dog collagen was detected in the moist black, yellow and red mural painting samples. This work was successful in using the ELISA method to detect the binding media in ancient Chinese mural paintings. ELISA can also be used for the rapid, direct detection of animal species in other ancient mural painting relics.